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Rapid Spectrophotometric Measurements of Total Bilirubin in Intact and Hemolyzed Neonatal Blood: A Feasibility Study

Journal of Perinatology volume 21, pages 382–387 (2001)Cite this article

Abstract

BACKGROUND: Conventional methods for measuring the total bilirubin concentration in blood require the use of serum or plasma, but physically separating red blood cells from plasma by centrifugation is a time-consuming and potentially dangerous process that does not lend itself to rapid, near-patient testing. Therefore, we have sought to determine whether spectrophotometric measurements of total bilirubin concentration are feasible in unaltered whole blood.

METHODS: We modified an Hb-Quick hemoglobinometer (Avox Systems, Inc., San Antonio, TX), a relatively new, portable, battery-powered instrument that uses disposable cuvettes and a reagentless system to measure total hemoglobin in nonhemolyzed, whole blood. The prototype consisted of an Hb-Quick equipped with light-emitting diodes with emissions at five different wavelengths to measure total bilirubin and total hemoglobin. Using blood samples from neonates with suspected hyperbilirubinemia, we made measurements on plasma from centrifuged samples and on hemolyzed and nonhemolyzed aliquots of the same blood samples.

RESULTS: In the first series of experiments, we compared the Unistat bilirubinometer's readings with the prototype's measurements of bilirubin in plasma. There was a close linear correlation between the prototype's measurements and those of the reference instrument (slope=1.01, r2=0.991). Subsequently, we used the prototype's measurements on plasma as the reference method and compared them with readings on hemolyzed and nonhemolyzed aliquots of each sample. Readings on hemolyzed and nonhemolyzed aliquots were significantly correlated with the measurements in plasma, but the regression lines did not have a slope of 1. However, when the measurements on hemolyzed and nonhemolyzed blood were scaled appropriately to compensate for the fact that red cells “dilute” the bilirubin in plasma, the correlation coefficients improved, and there was then a 1:1 relationship between the measurements in whole blood and plasma.

CONCLUSIONS: These preliminary findings demonstrate the feasibility of developing a portable instrument to measure total bilirubin in unaltered whole blood. The advantages of this method are speed, elimination of centrifugation or other sample preparation, and instrument portability. The disadvantage is that the concentration units are unconventional, i.e., milligrams of bilirubin per volume of whole blood. However, the instrument can be programmed to display the total bilirubin concentration in traditional units, e.g., milligrams of bilirubin per volume of plasma.

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Acknowledgements

The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NHLBI. The authors thank Brianne Backenstose for technical assistance and the nursing staff in the neonatal intensive care unit at University Hospital for help in obtaining the blood samples.

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Authors and Affiliations

  1. Department of Physiology, University of Texas Health Science Center, San Antonio, TX

    John M Steinke PhD & AP Shepherd PhD

  2. Department of Pediatrics, University of Texas Health Science Center, San Antonio, TX

    Alice K Gong MD

  3. Avox Systems, Inc., Fair Oaks Ranch, TX

    John M Steinke PhD & AP Shepherd PhD

Authors
  1. John M Steinke PhD
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  2. Alice K Gong MD
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  3. AP Shepherd PhD
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Additional information

This project was supported, in part, by grant 1R43 HL-64429 from the National Heart, Lung and Blood Institute.

APPENDIX

APPENDIX

To derive the relationship between the plasma and whole-blood concentrations of bilirubin, we begin with some simple definitions and conventions. Concentration is defined as the amount of bilirubin (Bil) divided by the fluid volume ( V) in which it is dispersed, and we let the subscripts p, rbc, and b denote plasma, red blood cells, and whole blood, respectively.

Equation 2 defines the bilirubin concentration in whole blood, but [Bil] b can be further defined as the sum of the amounts of bilirubin in red blood cells and plasma divided by total blood volume.

However, the amount of bilirubin inside red blood cells is negligible. Thus, Equation 6 becomes

Solving Equation 1 for the amount of bilirubin in plasma and substituting for Bil p in Equation 7 yields

From Equation 3 it can be deduced that the ratio of plasma volume to total blood volume ( Vp/ Vb) is 1−Hct, so substituting 1−Hct into Equation 8 gives

and solving for the bilirubin concentration in plasma, we obtain

Equations 9 and 10 make it readily apparent that the bilirubin concentration in whole blood can be converted into the concentration in plasma (and vice versa), if the hematocrit is known. Similarly, the ratio of total hemoglobin concentration to mean corpuscular hemoglobin concentration (Equation 5) can be used for the same purpose. Substituting [THb]/MCHC for Hct in Equation 10 and rearranging yields Equation 11.

Because the instrument measures both [Bil] b and [THb] in each sample of whole blood, Equations 10 and 11 illustrate how the bilirubin concentration in plasma is easily computed from the measurement in whole blood if either Hct or MCHC is known.

In the present study, we used the prototype's measurement of [THb] and an assumed MCHC of 34 g/dl to convert our measurements in whole blood into the plasma concentration. The error caused by assuming a constant MCHC varies with [THb]. Nevertheless, we can estimate the error introduced by the conversion if the actual MCHC is different from 34. For example, at a [THb] of 15 g/dl and a true [Bil] p of 16 mg/dl, the conversion would overestimate [Bil] p by 1.33 mg/dl (8%) at MCHC=31 and would underestimate [Bil] p by 0.97 mg/dl (6%) at MCHC=37. Fortunately, MCHC varies relatively little in healthy newborns; the standard deviation about the mean is only ±1.65 g/dl. 9 Of course, this SD indicates that 95% have MCHC values within ±3.3 g/dl of the mean.

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Steinke, J., Gong, A. & Shepherd, A. Rapid Spectrophotometric Measurements of Total Bilirubin in Intact and Hemolyzed Neonatal Blood: A Feasibility Study. J Perinatol 21, 382–387 (2001). https://doi.org/10.1038/sj.jp.7210547

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